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1.
J Lipid Res ; 64(4): 100351, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36868360

RESUMEN

Sphingosine 1-phosphate lyase (SGPL1) insufficiency (SPLIS) is a syndrome which presents with adrenal insufficiency, steroid-resistant nephrotic syndrome, hypothyroidism, neurological disease, and ichthyosis. Where a skin phenotype is reported, 94% had abnormalities such as ichthyosis, acanthosis, and hyperpigmentation. To elucidate the disease mechanism and the role SGPL1 plays in the skin barrier we established clustered regularly interspaced short palindromic repeats-Cas9 SGPL1 KO and a lentiviral-induced SGPL1 overexpression (OE) in telomerase reverse-transcriptase immortalised human keratinocytes (N/TERT-1) and thereafter organotypic skin equivalents. Loss of SGPL1 caused an accumulation of S1P, sphingosine, and ceramides, while its overexpression caused a reduction of these species. RNAseq analysis showed perturbations in sphingolipid pathway genes, particularly in SGPL1_KO, and our gene set enrichment analysis revealed polar opposite differential gene expression between SGPL1_KO and _OE in keratinocyte differentiation and Ca2+ signaling genesets. SGPL1_KO upregulated differentiation markers, while SGPL1_OE upregulated basal and proliferative markers. The advanced differentiation of SGPL1_KO was confirmed by 3D organotypic models that also presented with a thickened and retained stratum corneum and a breakdown of E-cadherin junctions. We conclude that SPLIS associated ichthyosis is a multifaceted disease caused possibly by sphingolipid imbalance and excessive S1P signaling, leading to increased differentiation and an imbalance of the lipid lamellae throughout the epidermis.


Asunto(s)
Ictiosis , Esfingolípidos , Humanos , Calcio/metabolismo , Aldehído-Liasas/genética , Aldehído-Liasas/metabolismo , Lisofosfolípidos/metabolismo , Esfingosina/genética , Esfingosina/metabolismo , Ictiosis/genética
2.
Int J Mol Sci ; 20(14)2019 Jul 12.
Artículo en Inglés | MEDLINE | ID: mdl-31336867

RESUMEN

BACKGROUND: Cutaneous squamous cell carcinoma (cSCC) incidence continues to rise with increasing morbidity and mortality, with limited treatment options for advanced disease. Future improvements in targeted therapy will rely on advances in genomic/transcriptomic understanding and the use of model systems for basic research. We describe here the panel of 16 primary and metastatic cSCC cell lines developed and characterised over the past three decades in our laboratory in order to provide such a resource for future preclinical research and drug screening. METHODS: Primary keratinocytes were isolated from cSCC tumours and metastases, and cell lines were established. These were characterised using short tandem repeat (STR) profiling and genotyped by whole exome sequencing. Multiple in vitro assays were performed to document their morphology, growth characteristics, migration and invasion characteristics, and in vivo xenograft growth. RESULTS: STR profiles of the cSCC lines allow the confirmation of their unique identity. Phylogenetic trees derived from exome sequence analysis of the matched primary and metastatic lines provide insight into the genetic basis of disease progression. The results of in vivo and in vitro analyses allow researchers to select suitable cell lines for specific experimentation. CONCLUSIONS: There are few well-characterised cSCC lines available for widespread preclinical experimentation and drug screening. The described cSCC cell line panel provides a critical tool for in vitro and in vivo experimentation.


Asunto(s)
Antineoplásicos/farmacología , Carcinoma de Células Escamosas/patología , Modelos Animales de Enfermedad , Evaluación Preclínica de Medicamentos/métodos , Neoplasias Cutáneas/patología , Animales , Biomarcadores de Tumor , Biopsia , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/metabolismo , Línea Celular Tumoral , Perfilación de la Expresión Génica , Humanos , Inmunohistoquímica , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Masculino , Mutación , Metástasis de la Neoplasia , Estadificación de Neoplasias , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Int J Mol Sci ; 19(4)2018 Mar 27.
Artículo en Inglés | MEDLINE | ID: mdl-29584680

RESUMEN

Background: Chronic skin wounds are a growing financial burden for healthcare providers, causing discomfort/immobility to patients. Whilst animal chronic wound models have been developed to allow for mechanistic studies and to develop/test potential therapies, such systems are not good representations of the human chronic wound state. As an alternative, human chronic wound fibroblasts (CWFs) have permitted an insight into the dysfunctional cellular mechanisms that are associated with these wounds. However, such cells strains have a limited replicative lifespan and therefore a limited reproducibility/usefulness. Objectives: To develop/characterise immortalised cell lines of CWF and patient-matched normal fibroblasts (NFs). Methods and Results: Immortalisation with human telomerase resulted in both CWF and NF proliferating well beyond their replicative senescence end-point (respective cell strains senesced as normal). Gene expression analysis demonstrated that, whilst proliferation-associated genes were up-regulated in the cell lines (as would be expected), the immortalisation process did not significantly affect the disease-specific genotype. Immortalised CWF (as compared to NF) also retained a distinct impairment in their wound repopulation potential (in line with CWF cell strains). Conclusions: These novel CWF cell lines are a credible animal alternative and could be a valuable research tool for understanding both the aetiology of chronic skin wounds and for therapeutic pre-screening.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Fibroblastos/citología , Modelos Biológicos , Enfermedades de la Piel/patología , Telomerasa/metabolismo , Experimentación Animal , Proliferación Celular , Células Cultivadas , Senescencia Celular , Enfermedad Crónica , Fibroblastos/química , Fibroblastos/patología , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Fenotipo , Enfermedades de la Piel/genética , Cicatrización de Heridas
4.
J Pathol ; 235(4): 581-92, 2015 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-25408555

RESUMEN

Biomechanical strain imposed by age-related thickening of the basal lamina and augmented tissue stiffness in the prostate gland coincides with increased cancer risk. Here we hypothesized that the structural alterations in the basal lamina associated with age can induce mechanotransduction pathways in prostate epithelial cells (PECs) to promote invasiveness and cancer progression. To demonstrate this, we developed a 3D model of PEC acini in which thickening and stiffening of basal lamina matrix was induced by advanced glycation end-product (AGE)-dependent non-enzymatic crosslinking of its major components, collagen IV and laminin. We used this model to demonstrate that antibody targeted blockade of CTLD2, the second of eight C-type lectin-like domains in Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) that can recognize glycosylated collagens, reversed actinomyosin-based contractility [myosin-light chain-2 (MLC2) phosphorylation], loss of cell polarity, loss of cell-cell junctions, luminal infiltration and basal invasion induced by AGE-modified basal lamina matrix in PEC acini. Our in vitro results were concordant with luminal occlusion of acini in the prostate glands of adult Endo180(Δ) (Ex2-6/) (Δ) (Ex2-6) mice, with constitutively exposed CTLD2 and decreased survival of men with early (non-invasive) prostate cancer with high epithelial Endo180 expression and levels of AGE. These findings indicate that AGE-dependent modification of the basal lamina induces invasive behaviour in non-transformed PECs via a molecular mechanism linked to cancer progression. This study provides a rationale for targeting CTLD2 in Endo180 in prostate cancer and other pathologies in which increased basal lamina thickness and tissue stiffness are driving factors. © 2014 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.


Asunto(s)
Membrana Basal/metabolismo , Movimiento Celular , Células Epiteliales/metabolismo , Productos Finales de Glicación Avanzada/metabolismo , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Mitogénicos/metabolismo , Animales , Membrana Basal/patología , Línea Celular Tumoral , Supervivencia Celular , Elasticidad , Humanos , Estimación de Kaplan-Meier , Lectinas Tipo C/metabolismo , Masculino , Mecanotransducción Celular , Glicoproteínas de Membrana/deficiencia , Glicoproteínas de Membrana/genética , Ratones Noqueados , Invasividad Neoplásica , Neoplasias de la Próstata/mortalidad , Neoplasias de la Próstata/patología , Estructura Terciaria de Proteína , Receptores de Superficie Celular/deficiencia , Receptores de Superficie Celular/genética , Factores de Tiempo
5.
Nucleic Acids Res ; 41(10): 5400-12, 2013 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-23580553

RESUMEN

Uncontrolled cell proliferation and cytoskeletal remodeling are responsible for tumor development and ultimately metastasis. A number of studies have implicated microRNAs in the regulation of cancer cell invasion and migration. Here, we show that miR-23b regulates focal adhesion, cell spreading, cell-cell junctions and the formation of lamellipodia in breast cancer (BC), implicating a central role for it in cytoskeletal dynamics. Inhibition of miR-23b, using a specific sponge construct, leads to an increase of cell migration and metastatic spread in vivo, indicating it as a metastatic suppressor microRNA. Clinically, low miR-23b expression correlates with the development of metastases in BC patients. Mechanistically, miR-23b is able to directly inhibit a number of genes implicated in cytoskeletal remodeling in BC cells. Through intracellular signal transduction, growth factors activate the transcription factor AP-1, and we show that this in turn reduces miR-23b levels by direct binding to its promoter, releasing the pro-invasive genes from translational inhibition. In aggregate, miR-23b expression invokes a sophisticated interaction network that co-ordinates a wide range of cellular responses required to alter the cytoskeleton during cancer cell motility.


Asunto(s)
Neoplasias de la Mama/genética , Neoplasias de la Mama/patología , Movimiento Celular , Citoesqueleto/metabolismo , Regulación Neoplásica de la Expresión Génica , MicroARNs/metabolismo , Animales , Neoplasias de la Mama/metabolismo , Miosinas Cardíacas/metabolismo , Adhesión Celular , Línea Celular , Línea Celular Tumoral , Movimiento Celular/genética , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Femenino , Adhesiones Focales/ultraestructura , Humanos , Ratones , Ratones Desnudos , MicroARNs/antagonistas & inhibidores , MicroARNs/genética , Cadenas Ligeras de Miosina/metabolismo , Metástasis de la Neoplasia , Fosforilación , Regiones Promotoras Genéticas , Seudópodos/ultraestructura , Factor de Transcripción AP-1/metabolismo , Transcripción Genética , Quinasas p21 Activadas/metabolismo
6.
J Breast Imaging ; 6(3): 277-287, 2024 May 27.
Artículo en Inglés | MEDLINE | ID: mdl-38537570

RESUMEN

OBJECTIVE: We investigated patient experience with screening contrast-enhanced mammography (CEM) to determine whether a general population of women with dense breasts would accept CEM in a screening setting. METHODS: In this institutional review board-approved prospective study, patients with heterogeneous and extremely dense breasts on their mammogram were invited to undergo screening CEM and complete pre-CEM and post-CEM surveys. On the pre-CEM survey, patients were asked about their attitudes regarding supplemental screening in general. On the post-CEM survey, patients were asked about their experience undergoing screening CEM, including causes and severity of any discomfort and whether they would consider undergoing screening CEM again in the future or recommend it to a friend. RESULTS: One hundred sixty-three women were surveyed before and after screening CEM. Most patients, 97.5% (159/163), reported minimal or no unpleasantness associated with undergoing screening CEM. In addition, 91.4% (149/163) said they would probably or very likely undergo screening CEM in the future if it cost the same as a traditional screening mammogram, and 95.1% (155/163) said they would probably or very likely recommend screening CEM to a friend. Patients in this study, who were all willing to undergo CEM, more frequently reported a family history of breast cancer than a comparison cohort of women with dense breasts (58.2% vs 47.1%, P = .027). CONCLUSION: Patients from a general population of women with dense breasts reported a positive experience undergoing screening CEM, suggesting screening CEM might be well received by this patient population, particularly if the cost was comparable with traditional screening mammography.


Asunto(s)
Densidad de la Mama , Neoplasias de la Mama , Medios de Contraste , Mamografía , Humanos , Femenino , Mamografía/métodos , Persona de Mediana Edad , Neoplasias de la Mama/diagnóstico por imagen , Neoplasias de la Mama/diagnóstico , Medios de Contraste/administración & dosificación , Estudios Prospectivos , Anciano , Adulto , Detección Precoz del Cáncer/métodos , Encuestas y Cuestionarios , Mama/diagnóstico por imagen , Intensificación de Imagen Radiográfica/métodos , Tamizaje Masivo/métodos
7.
J Invest Dermatol ; 2024 Aug 09.
Artículo en Inglés | MEDLINE | ID: mdl-39127929

RESUMEN

Skin in vitro models offer much promise for research, testing drugs, cosmetics, and medical devices, reducing animal testing and extensive clinical trials. There are several in vitro approaches to mimicking human skin behavior, ranging from simple cell monolayer to complex organotypic and bioengineered 3-dimensional models. Some have been approved for preclinical studies in cosmetics, pharmaceuticals, and chemicals. However, development of physiologically reliable in vitro human skin models remains in its infancy. This review reports on advances in in vitro complex skin models to study skin homeostasis, aging, and skin disease.

8.
Cell Tissue Res ; 351(2): 255-68, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-22526628

RESUMEN

Epidermal wound healing is a complex and highly coordinated process where several different cell types and molecules, such as growth factors and extracellular matrix (ECM) components, play an important role. Among the many proteins that are essential for the restoration of tissue integrity is the metalloproteinase (MMP) family. MMPs can act on ECM and non-ECM components affecting degradation and modulation of the ECM, growth-factor activation and cell-cell and cell-matrix signalling. MMPs are secreted by different cell types such as keratinocytes, fibroblasts and inflammatory cells at different stages and locations during wound healing, thereby regulating this process in a very coordinated and controlled way. In this article, we review the role of MMPs and their inhibitors (TIMPs), as well as the disintegrin and metalloproteinase with the thrombospondin motifs (ADAMs) family, in epithelial wound repair.


Asunto(s)
Epidermis/enzimología , Metaloproteinasas de la Matriz/metabolismo , Cicatrización de Heridas/fisiología , Animales , Células Epidérmicas , Epidermis/metabolismo , Humanos , Queratinocitos/citología , Queratinocitos/enzimología , Metaloproteinasas de la Matriz/fisiología
9.
J Pathol ; 226(5): 775-83, 2012 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-22072289

RESUMEN

Cellular niches in adult tissue can harbour dysregulated microenvironments that become the driving force behind disease progression. The major environmental change when metastatic cells arrive in the bone is the destruction of mineralized type I collagen matrix. Once metastatic niches establish in bone, the invading tumour cells initiate a vicious cycle of osteolytic lesion formation via the dysregulation of paracrine signals and uncoupling of normal bone resorption and production. Here we report that the collagen receptor Endo180 (CD280, MRC2, uPARAP) participates in collagen deposition by primary human osteoblasts during de novo osteoid formation. This newly recognized function of Endo180 was suppressed in osteoblasts following heterotypic direct cell-cell contact in co-culture with prostate tumour cells. Reciprocal Endo180 up-regulation in osteolytic prostate tumour cells (PC3 and DU145) followed their direct contact with osteoblasts and promoted de novo collagen internalization, which is a previously characterized function of the constitutively recycling Endo180 receptor. The osteoblastic suppression and tumour cell-associated enhancement of Endo180 expression were equally sustained in these direct co-cultures. These findings are the first to demonstrate that increased tumour cell participation in collagen degradation and decreased collagen formation by osteoblasts in the osteolytic microenvironment are linked to the divergent regulation of a collagen-binding receptor. Immunohistochemical analysis of core biopsies from bone metastasis revealed higher levels of Endo180 expression in tumour cell foci than cells in the surrounding stroma. Additional experiments in prostate cell-osteoblast co-cultures indicate that divergent regulation of Endo180 is the result of dysregulated TGFß1 signalling. The findings of this study provide a rationale for targeting collagen remodelling by Endo180 in bone metastases and other collagen matrix pathologies.


Asunto(s)
Neoplasias Óseas/metabolismo , Colágeno Tipo I/metabolismo , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Osteoblastos/metabolismo , Neoplasias de la Próstata/metabolismo , Receptores de Superficie Celular/metabolismo , Receptores Mitogénicos/metabolismo , Células del Estroma/metabolismo , Factor de Crecimiento Transformador beta1/metabolismo , Comunicación Autocrina , Biopsia , Neoplasias Óseas/genética , Neoplasias Óseas/secundario , Línea Celular Tumoral , Técnicas de Cocultivo , Técnica del Anticuerpo Fluorescente , Humanos , Inmunohistoquímica , Masculino , Lectinas de Unión a Manosa/genética , Glicoproteínas de Membrana/genética , Osteoblastos/efectos de los fármacos , Osteoblastos/patología , Comunicación Paracrina , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/patología , Inhibidores de Proteínas Quinasas/farmacología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/metabolismo , Interferencia de ARN , Receptor Tipo I de Factor de Crecimiento Transformador beta , Receptores de Superficie Celular/genética , Receptores Mitogénicos/genética , Receptores de Factores de Crecimiento Transformadores beta/antagonistas & inhibidores , Receptores de Factores de Crecimiento Transformadores beta/metabolismo , Transducción de Señal , Células del Estroma/efectos de los fármacos , Células del Estroma/patología , Transfección , Microambiente Tumoral
10.
Front Mol Biosci ; 10: 1176802, 2023.
Artículo en Inglés | MEDLINE | ID: mdl-37363400

RESUMEN

Recessive X-linked ichthyosis (RXLI), a genetic disorder caused by deletion or point mutations of the steroid sulfatase (STS) gene, is the second most common form of ichthyosis. It is a disorder of keratinocyte cholesterol sulfate retention and the mechanism of extracutaneous phenotypes such as corneal opacities and attention deficit hyperactivity disorder are poorly understood. To understand the pathomechanisms of RXLI, the transcriptome of differentiated primary keratinocytes with STS knockdown was sequenced. The results were validated in a stable knockdown model of STS, to confirm STS specificity, and in RXLI skin. The results show that there was significantly reduced expression of genes related to epidermal differentiation and lipid metabolism, including ceramide and sphingolipid synthesis. In addition, there was significant downregulation of aldehyde dehydrogenase family members and the oxytocin receptor which have been linked to corneal transparency and behavioural disorders respectively, both of which are extracutaneous phenotypes of RXLI. These data provide a greater understanding of the causative mechanisms of RXLI's cutaneous phenotype, and show that the keratinocyte transcriptome and lipidomics can give novel insights into the phenotype of patients with RXLI.

11.
Nat Struct Mol Biol ; 30(4): 527-538, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-37012406

RESUMEN

The placenta is a fast-evolving organ with large morphological and histological differences across eutherians, but the genetic changes driving placental evolution have not been fully elucidated. Transposable elements, through their capacity to quickly generate genetic variation and affect host gene regulation, may have helped to define species-specific trophoblast gene expression programs. Here we assess the contribution of transposable elements to human trophoblast gene expression as enhancers or promoters. Using epigenomic data from primary human trophoblast and trophoblast stem-cell lines, we identified multiple endogenous retrovirus families with regulatory potential that lie close to genes with preferential expression in trophoblast. These largely primate-specific elements are associated with inter-species gene expression differences and are bound by transcription factors with key roles in placental development. Using genetic editing, we demonstrate that several elements act as transcriptional enhancers of important placental genes, such as CSF1R and PSG5. We also identify an LTR10A element that regulates ENG expression, affecting secretion of soluble endoglin, with potential implications for preeclampsia. Our data show that transposons have made important contributions to human trophoblast gene regulation, and suggest that their activity may affect pregnancy outcomes.


Asunto(s)
Retrovirus Endógenos , Trofoblastos , Animales , Humanos , Embarazo , Femenino , Trofoblastos/metabolismo , Placenta/metabolismo , Retrovirus Endógenos/genética , Elementos Transponibles de ADN/genética , Regulación de la Expresión Génica , Expresión Génica
12.
Antioxidants (Basel) ; 12(7)2023 Jun 30.
Artículo en Inglés | MEDLINE | ID: mdl-37507914

RESUMEN

Unlike skin, oral mucosal wounds are characterized by rapid healing and minimal scarring, attributable to the "enhanced" healing properties of oral mucosal fibroblasts (OMFs). As oxidative stress is increasingly implicated in regulating wound healing outcomes, this study compared oxidative stress biomarker and enzymic antioxidant profiles between patient-matched oral mucosal/skin tissues and OMFs/skin fibroblasts (SFs) to determine whether superior oral mucosal antioxidant capabilities and reduced oxidative stress contributed to these preferential healing properties. Oral mucosa and skin exhibited similar patterns of oxidative protein damage and lipid peroxidation, localized within the lamina propria/dermis and oral/skin epithelia, respectively. SOD1, SOD2, SOD3 and catalase were primarily localized within epithelial tissues overall. However, SOD3 was also widespread within the lamina propria localized to OMFs, vasculature and the extracellular matrix. OMFs were further identified as being more resistant to reactive oxygen species (ROS) generation and oxidative DNA/protein damage than SFs. Despite histological evaluation suggesting that oral mucosa possessed higher SOD3 expression, this was not fully substantiated for all OMFs examined due to inter-patient donor variability. Such findings suggest that enzymic antioxidants have limited roles in mediating privileged wound healing responses in OMFs, implying that other non-enzymic antioxidants could be involved in protecting OMFs from oxidative stress overall.

13.
J Breast Imaging ; 4(1): 19-24, 2022.
Artículo en Inglés | MEDLINE | ID: mdl-35103252

RESUMEN

OBJECTIVE: Some vendors have created algorithms that generate synthetic 2D (s2D) images from a digital breast tomosynthesis (DBT) dataset to reduce the radiation from obtaining a separate 2D digital mammography (DM). This study evaluated the visibility of amorphous calcifications on 2D DM versus s2D on screening mammography. METHODS: This IRB-approved, retrospective, reader study included screening mammograms from 36 women who received screening DBT exams where both 2D DM and s2D images were obtained: 28 screening mammograms that were eventually given BI-RADS category 4 or 5 for amorphous calcifications and 8 BI-RADS category 1 or 2 screening exams. Two rounds of interpretation were conducted with a six-week washout period. Cases were randomized to display either the 2D DM or s2D images, which were then alternated in the second round. Four fellowship-trained breast radiologists determined whether a study merited recall for calcifications. If so, they rated calcification visibility on a scale of 1 to 5. McNemar chi-square tests were conducted to assess differences in recall rates and Wilcoxon signed rank tests were used to examine shifts in visibility. RESULTS: There was no difference in detection rates of amorphous calcifications between 2D DM and s2D, which were 75.9% and 75.0%, respectively (P = 1.000). Collectively, amorphous calcifications were more visible on s2D than 2D DM, with mean visibility scores of 3.4 versus 3.0, respectively (P = 0.005). CONCLUSION: Synthetic 2D did not change identification of amorphous calcifications compared to 2D DM, and readers considered them more visible on average.

14.
J Dent ; 125: 104251, 2022 10.
Artículo en Inglés | MEDLINE | ID: mdl-35961474

RESUMEN

OBJECTIVES: The gingiva heals at an accelerated rate with reduced scarring when compared to skin. Potential well-studied factors include immune cell number, angiogenesis disparities and fibroblast gene expression. Differential keratinocyte gene expression, however, remains relatively understudied. This study explored the contrasting healing efficiencies of gingival and skin keratinocytes, alongside their differential gene expression patterns. METHODS: 3D organotypic culture models of human gingiva and skin were developed using temporarily immortalised primary keratinocytes. Models were wounded for visualisation of re-epithelialisation and analysis of keratinocyte migration to close the wound gap. Concurrently, differentially expressed genes between primary gingival and skin keratinocytes were identified, validated, and functionally assessed. RESULTS: Characterisation of the 3D cultures of gingiva and skin showed differentiation markers that recapitulated organisation of the corresponding in vivo tissue. Upon wounding, gingival models displayed a significantly higher efficiency in re-epithelialisation and stratification versus skin, repopulating the wound gap within 24 hours. This difference was likely due to distinct patterns of migration, with gingival cells demonstrating a form of sheet migration, in contrast to skin, where the leading edge was typically 1-2 cells thick. A candidate approach was used to identify several genes that were differentially expressed between gingival and skin keratinocytes. Knockdown of PITX1 resulted in reduced migration capacity of gingival cells. CONCLUSION: Gingival keratinocytes retain in vivo superior wound healing capabilities in in vitro 2D and 3D environments. Intrinsic gene expression differences could result in gingival cells being 'primed' for healing and play a role in faster wound resolution. CLINICAL SIGNIFICANCE STATEMENT: The successful development of organotypic models, that recapitulate re-epithelialisation, will underpin further studies to analyse the oral response to wound stimuli, and potential therapeutic interventions, in an in vitro environment.


Asunto(s)
Encía , Queratinocitos , Células Cultivadas , Fibroblastos , Humanos , Queratinocitos/metabolismo , Piel/lesiones , Piel/metabolismo , Cicatrización de Heridas/fisiología
15.
J Clin Invest ; 130(9): 4798-4810, 2020 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-32544098

RESUMEN

The biology of harlequin ichthyosis (HI), a devastating skin disorder caused by loss-of-function mutations in the gene ABCA12, is poorly understood, and to date, no satisfactory treatment has been developed. We sought to investigate pathomechanisms of HI that could lead to the identification of new treatments for improving patients' quality of life. In this study, RNA-Seq and functional assays were performed to define the effects of loss of ABCA12 using HI patient skin samples and an engineered CRISPR/Cas9 ABCA12 KO cell line. The HI living skin equivalent (3D model) recapitulated the HI skin phenotype. The cytokines IL-36α and IL-36γ were upregulated in HI skin, whereas the innate immune inhibitor IL-37 was strongly downregulated. We also identified STAT1 and its downstream target inducible nitric oxide synthase (NOS2) as being upregulated in the in vitro HI 3D model and HI patient skin samples. Inhibition of NOS2 using the inhibitor 1400W or the JAK inhibitor tofacitinib dramatically improved the in vitro HI phenotype by restoring the lipid barrier in the HI 3D model. Our study has identified dysregulated pathways in HI skin that are feasible therapeutic targets.


Asunto(s)
Amidinas/farmacología , Bencilaminas/farmacología , Sistemas de Liberación de Medicamentos , Ictiosis Lamelar , Modelos Biológicos , Óxido Nítrico Sintasa de Tipo II/antagonistas & inhibidores , Piperidinas/farmacología , Pirimidinas/farmacología , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Técnicas de Cultivo de Célula , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Ictiosis Lamelar/tratamiento farmacológico , Ictiosis Lamelar/genética , Ictiosis Lamelar/metabolismo , Ictiosis Lamelar/patología , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/metabolismo , Inflamación/patología , Interleucina-1/genética , Interleucina-1/metabolismo , Mutación con Pérdida de Función , Óxido Nítrico Sintasa de Tipo II/genética , Óxido Nítrico Sintasa de Tipo II/metabolismo , Factor de Transcripción STAT1/genética , Factor de Transcripción STAT1/metabolismo
16.
Genes Chromosomes Cancer ; 47(3): 238-46, 2008 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-18069666

RESUMEN

Telomerase activity (TA) and the expression of its enzymatic subunits, which have been demonstrated in many tumors, remain poorly investigated in tumors associated with neurofibromatosis type 1 (NF1). In this study, we analysed the association of TA and the expression of telomerase RNA (TR) and telomerase reverse transcriptase (TERT) in 23 malignant peripheral nerve sheath tumors (MPNST) (17 high grade and 6 low grade tumors), 11 plexiform neurofibromas (PNF) and 6 dermal neurofibromas (DNF). TA was studied using telomerase repeat amplification protocol (TRAP) assay and expression of TR and TERT was investigated using reverse transcription PCR (RT-PCR) and real-time PCR. TA was detected in 14 out of 17 (82%) high grade MPNST, whereas all 6 low grade MPNST and 17 benign tumors were telomerase negative. The TERT transcripts were detected in all high grade MPNST, 50% of the low grade MPNST, and 4 benign tumors. However, the expression level of the TERT strikingly correlated with TA and high grade MPNST. Thus, while TERT expression was similar in both low grade MPNST and PNF (P = 0.115), it was significantly higher in high grade MPNST when compared to either low grade MPNST (P = 0.042), PNF (P = 0.001) or DNF tumors (P = 0.010). These findings indicate that TA and expression level of TERT are potential markers for high grade malignancy in NF1 patients.


Asunto(s)
Neoplasias de la Vaina del Nervio/diagnóstico , Neurofibromatosis 1/complicaciones , ARN/análisis , Índice de Severidad de la Enfermedad , Telomerasa/análisis , Telomerasa/metabolismo , Biomarcadores/análisis , Humanos , Neoplasias de la Vaina del Nervio/etiología , Neurofibroma/diagnóstico , Neurofibroma Plexiforme/diagnóstico
18.
J Trauma Acute Care Surg ; 81(2): 229-35, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-27050881

RESUMEN

BACKGROUND: Pediatric trauma patients transferred to pediatric trauma centers (PTCs) often have imaging at the originating hospital (OH). The increased use of computed tomography (CT) raises concerns about malignancy risk from ionizing radiation leading many PTCs to adopt radiation dose reduction strategies. We hypothesized that pediatric trauma patients are exposed to excess radiation from imaging before transfer. METHODS: A retrospective review of 1,383 scans was performed on all trauma patients with CT imaging before transfer to our Level I PTC from 2010 to 2014. Demographics, type of imaging, necessity for repeat imaging, appropriateness of imaging, and radiation dose delivered were recorded. Comparative radiation dosing was calculated using the dose-length product (DLP [expressed in mGy-cm]). All CT scans except for CT of the abdomen and pelvis and CT of the head were excluded for complete DLP data issues. Scans were considered clinically appropriate if they met Advanced Trauma Life Support (ATLS) recommendations (ATLS+) and not indicated if they did not meet ATLS criteria (ATLS-). Some scans were repeated because of technical issues. Median ΔDLP represents the difference in dose patients received at OH versus at PTC. RESULTS: A total of 673 patients were analyzed. Average age was 11 years, and 65.4% were male. Mean DLP at PTC was 54% lower for all analyzed scans compared with OH (p < 0.0001). DLP at PTC was 51% lower for CT of the abdomen and pelvis and 62% lower for CT of the head. Children received excess dose of 578.62 mGy-cm for scans at OH that were unnecessary. For ATLS+ imaging, children received a median excess of 444.42 mGy-cm of radiation at OH than they would have received had the scans been performed at PTCs using pediatric radiation reduction strategies. CONCLUSION: Pediatric trauma imaging performed at transferring institutions often does not adhere to ATLS recommendations and exceeds required ionizing radiation dosages. This study further confirms ATLS recommendations supporting prompt patient transfer without delay for imaging. LEVEL OF EVIDENCE: Therapeutic study, level IV.


Asunto(s)
Transferencia de Pacientes , Dosis de Radiación , Tomografía Computarizada por Rayos X , Heridas y Lesiones/diagnóstico por imagen , Niño , Femenino , Hospitales Pediátricos , Humanos , Masculino , Estudios Retrospectivos , Centros Traumatológicos
19.
Dev Cell ; 36(2): 152-63, 2016 Jan 25.
Artículo en Inglés | MEDLINE | ID: mdl-26812015

RESUMEN

Critical roles for DNA methylation in embryonic development are well established, but less is known about its roles during trophoblast development, the extraembryonic lineage that gives rise to the placenta. We dissected the role of DNA methylation in trophoblast development by performing mRNA and DNA methylation profiling of Dnmt3a/3b mutants. We find that oocyte-derived methylation plays a major role in regulating trophoblast development but that imprinting of the key placental regulator Ascl2 is only partially responsible for these effects. We have identified several methylation-regulated genes associated with trophoblast differentiation that are involved in cell adhesion and migration, potentially affecting trophoblast invasion. Specifically, trophoblast-specific DNA methylation is linked to the silencing of Scml2, a Polycomb Repressive Complex 1 protein that drives loss of cell adhesion in methylation-deficient trophoblast. Our results reveal that maternal DNA methylation controls multiple differentiation-related and physiological processes in trophoblast via both imprinting-dependent and -independent mechanisms.


Asunto(s)
Diferenciación Celular/genética , Linaje de la Célula/genética , Metilación de ADN , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica/genética , Placenta/embriología , Trofoblastos/citología , Animales , Epigénesis Genética/genética , Femenino , Impresión Genómica/genética , Ratones Transgénicos , Placenta/metabolismo , Embarazo
20.
Clin Exp Metastasis ; 33(2): 151-65, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26567111

RESUMEN

The diverse composition and structure of extracellular matrix (ECM) interfaces encountered by tumor cells at secondary tissue sites can influence metastatic progression. Extensive in vitro and in vivo data has confirmed that metastasizing tumor cells can adopt different migratory modes in response to their microenvironment. Here we present a model that uses human stromal cell-derived matrices to demonstrate that plasticity in tumor cell movement is controlled by the tumor-associated collagen receptor Endo180 (CD280, CLEC13E, KIAA0709, MRC2, TEM9, uPARAP) and the crosslinking of collagen fibers by stromal-derived lysyl oxidase (LOX). Human osteoblast-derived and fibroblast-derived ECM supported a rounded 'amoeboid-like' mode of cell migration and enhanced Endo180 expression in three prostate cancer cell lines (PC3, VCaP, DU145). Genetic silencing of Endo180 reverted PC3 cells from their rounded mode of migration towards a bipolar 'mesenchymal-like' mode of migration and blocked their translocation on human fibroblast-derived and osteoblast-derived matrices. The concomitant decrease in PC3 cell migration and increase in Endo180 expression induced by stromal LOX inhibition indicates that the Endo180-dependent rounded mode of prostate cancer cell migration requires ECM crosslinking. In conclusion, this study introduces a realistic in vitro model for the study of metastatic prostate cancer cell plasticity and pinpoints the cooperation between tumor-associated Endo180 and the stiff microenvironment imposed by stromal-derived LOX as a potential target for limiting metastatic progression in prostate cancer.


Asunto(s)
Movimiento Celular , Matriz Extracelular/metabolismo , Lectinas de Unión a Manosa/metabolismo , Glicoproteínas de Membrana/metabolismo , Neoplasias de la Próstata/patología , Proteína-Lisina 6-Oxidasa/metabolismo , Receptores de Superficie Celular/metabolismo , Microambiente Tumoral/fisiología , Línea Celular Tumoral , Fibroblastos/metabolismo , Citometría de Flujo , Humanos , Immunoblotting , Técnicas In Vitro , Masculino , Invasividad Neoplásica , Osteoblastos/metabolismo , Células del Estroma/metabolismo
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